BIOB34 Tutorial - Glucose Metabolism PDF

Week 1 | Tutorial #1 | BIOB34 (Thursday, Sept. 5th, 5:10 PM) [Continuing Module 2] Glucose Metabolism Cycle can either start with: a. Glucose being imported from a cell (eg. from bloodstream) OR b. From glycogen Glycolysis: glucose breakdown ○ P...

Week 1 | Tutorial #1 | BIOB34 (Thursday, Sept. 5th, 5:10 PM) [Continuing Module 2] Glucose Metabolism Cycle can either start with: a. Glucose being imported from a cell (eg. from bloodstream) OR b. From glycogen Glycolysis: glucose breakdown ○ Produces reducing equivalents ○ Releases energy Glucose + 2ADP + 2NAD+ → 2ATP + 2 pyruvate + 2NADH + 2H+ ○ Takes place in cytoplasm ○ Does not require oxygen ○ Produces intermediates for synthesis of various molecules (eg. carbohydrates, nucleic acid, amino acids, and fatty acid) Pyruvate (the end product) can be used for other catabolic processes *Basically, the NADH in the equation above can donate itself to the electron chain, but since it’s in the cytoplasm, it has to find a way to get in the mitochondria (PDH is inside of the mitochondria, we’ll talk about that soon) Pyruvate is cool, but it can be cooler with oxygen wow! ↓ Oxidation of Pyruvate in the Presence of O2 Glycolysis ○ Converts carbohydrates to pyruvate within the cytoplasm ○ Lactate and amino acids can also be converted to pyruvate ○ Pyruvate is carried into mitochondria When the pyruvate gets into the mitochondria, it gets with pyruvate dehydrogenase (PDH) Pyruvate dehydrogenase (PDH) (inside the mitochondria) ○ Pyruvate is oxidized by PDH to form acetyl CoA + NADH (which can be used for Krebs cycle) Oxidation of NADH in the Presence of O2 The NADH made from glycolysis can donate its energy to the electron transport chain BUT the electron chain is in the mitochondria, whereas the NADH is in the cytoplasm  Glycolysis can only continue if NADH is oxidized to NAD+ ○ Aka glycolysis outputs NADH, but needs NAD+ to continue (there’s a limited quantity of NAD+) ○ So what can we do with the extra NADH? We can give it to the mitochondria where it’ll use it up ○ How do we get NADH from the cytoplasm into the mitochondria? There’s two “shuttles” for it (these carry reducing equivalents from cytoplasm into mitochondria): α-glycerophosphate shuttle (seen in invertebrate animals) Malate-aspartate shuttle (see in mammals + most vertebrae) Oxidation of NADH in the Absence of O2 It’s possible to ramp up glycolysis (at least for a short time) without reducing agency in the mitochondria NADH cannot be used rapidly by mitochondria when oxygen is not present ○ NADH is oxidized in the cytoplasm THE STORY SO FAR: Ok, so, glucose outputs NADH and pyruvate. When the NADH goes into the mitochondria, it gets oxidized, turns back into NAD+, and in the process, can make all sorts of cool stuff. If it can’t go into the mitochondria (due to lack of oxygen), how will we be able to provide NAD+ for glycolysis? Introducing… LACTATE! Humans can produce lactate from pyruvate buildup (lactate = energy) Pyruvate + NADH + H+ ↔ lactate + NAD+ ○ Catalyzed by the enzyme lactate dehydrogenase (LDH) LDH can catalyze both ways (it’s cool and it helps react fast) So what happens when you get access to oxygen again? Lactate can turn back into pyruvate and NADH (along with the new oxygen) can get back into the mitochondria! Other anaerobic pathways form less toxic end products and more ATP than lactate (2 ATP) ○ Eg. succinate (4 ATP) and propionate (6 ATP) ○ More common in invertebrates (we can’t make these compounds) Lipids All are hydrophobic (do not dissolve in water) Carbon backbone  ○ Aliphatic: linear arrangement ○ Aromatic: ring arrangement ○ Examples: farry acids, triglycerides, phospholipids, steroids Lipids are used for energy metabolism, cell structure (eg. membranes), and signalling Fatty Acids Chain of carbon atoms ending with a carboxyl group ○ Usually an even number of carbons Saturated ○ No double bonds between carbons Unsaturated ○ One or more double bonds between carbons Fatty Acid Oxidation (β-Oxidation) Use this to make use of lipids for energy Fatty acids are more dense form of energy storage than carbohydrates ○ Water associates with carbs, not with oils/fats More reduced form of carbon ○ Take more O2 (more oxidation) to unlock energy ○ No significant anaerobic ATP production possible Ketones Some tissues cannot metabolize fatty acids, but they can metabolize ketones ○ For example, vertebrate brain, shark muscle Mitochondrial (Oxidative) Metabolism Some amino acids can also be utilized for energy production Energy-yielding reactions that require oxygen ○ Enzymes convert nutrients into metabolites ○ Metabolites enter mitochondria ○ Many metabolites are converted to acetyl CoA (in mitochondria) ○ Acetyl CoA enters the tricarboxylic acid cycle (The citric acid cycle/Krebs cycle) ○ Acetyl CoA is oxidized to form reducing equivalents ○ Reducing equivalents are oxidized to release energy ○ O2 is final electron acceptor Oxygen and Animals All animals depend on O2 EXCEPT a parasite called “cnidarian parasite” ○ Parasite lacked a mitochondrial genome ○ They don’t have the machinery for them to rely on oxygen  ○ Since it lives inside the salmon, it can just absorb the nutrients from the muscle (yummy body soup using glycolysis) ○ They outsource most of their functions onto the animal Oxidative Metabolism Acetyl CoA → Tricarboxylic acid (TCA) cycle: CO2 + reducing equivalent (NADH and FADH2) and GTP → Electron transport system (ETS): reducing equivalents are oxidized to release energy → Oxidative phosphorylation: ATP synthesis (phosphorylation) Tricarboxylic Acid (TCA) Cycle Generates reducing equivalents within the mitochondria Acetyl CoA + 3NAD+ + GDP + Pi + FAD → 2CO2 + 3NADH + FADH2 + GTP Amphibolic pathway (both anabolic and catabolic) ○ Some intermediates are broken down (catabolic) ○ Some intermediates are used for syntheses (anabolic) Electoral Transport System (ETS) Electrons from NADH and FADH2 are transferred to the ETS ○ Found within the inner mitochondrial membrane ○ Composed of four multisubunit proteins (complexes I, II, III, IV) and two electron carriers (ubiquinone and cytochrome c) ○ Oxidation: 4e- + 4H+ + O2 → 2H2O ○ Generates a proton gradient, heat, water, and reactive oxygen species (ROS) ATP Synthesis Phosphorylation: ○ ADP + Pi → ATP Proton motive force (Δp) ○ pH gradient and the membrane potential (ΔΨ) F1F0ATPase uses energy in Δp to produce ATP There is no physical linkage between oxidation and phosphorylation Two processes are functionally coupled through Δp Phosphocreatine Alternate high-energy phosphate-containing molecule Creatine + ATP ↔ ADP + phosphocreatine ○ Creatine phosphokinase (CPK)  Reaction is reversible so relative rate of ATP vs. phosphocreatine production depends on ratio of concentration of substrates/products Phosphocreatine can also move through the cell (like ATP) ○ Thus, it can enhance flux of high energy phosphate molecules from site of synthesis (eg. mitochondria) to site of hydrolysis (eg. muscle sarcomeres) Integration of Metabolic Pathways Gluconeogenesis: process transforming non-carbohydrate substrates into glucose ○ Happens in the liver mainly Fluctuations in nutrient availability energy demand, and environmental conditions Reciprocal regulation avoids simultaneous synthesis and degradation (futile cycles) Use of appropriate metabolic “fuel” ○ Carbohydrate vs lipid Measuring Metabolic Rate: 31P-NMR Spectroscopy Measures ATP turnover (NOT ATP consumption because homeostasis needs specific ATP concentration) ○ Detects change in NMR spectra as Pi groups shift between ATP and inorganic phosphate Pros ○ Measures cellular energy currency (accounts for aerobic, anaerobic, metabolism) ○ Accurate over very short time scales (eg. a single muscle contraction) Cons ○ Logistically difficult ○ Subject must be restrained, possibly anesthetized *Heat is a byproduct of all catabolic (and anabolic) steps Hess’s Law Hess’s Law: ○ Total amount of energy released (eventually as heat) for breakdown of given amount of fuel always the same Regardless of intermediate chemical steps (eg. particular ATP synthesis pathway) ○ Aka more heat = more energy Measuring Metabolic Rate: Direct Calorimetry Calorimetry: measurement of health of chemical/physiological processes (unit: ‘calorie’) Pros:  ○ Quite accurate under many conditions ○ Accounts for aerobic anaerobic energy production Cons: ○ Subject must be restrained ○ Equipment heavy and complicated ○ Makes assumptions about anabolic vs catabolic activity How it works: 1. Animal held in central chamber surrounded by two concentric chambers filled with ice or ice water 2. Exterior of the 2 is to buffer influx of heat from outside ○ Melts ice, but inner edge stays ice old 3. Interior of the 2 melts due to animal heat production only 4. Water collected + volume measured Calculation of metabolic rate can be done if we know how much heat it takes to melt a given amount of ice Measuring Metabolic Rate: Indirect Calorimetry Indirect calorimetry ○ Inferring metabolic heat production (eg. through respiratory gas exchange - respirometry) Pros: ○ Pretty user friendly and easy ○ Equipment can be portable ○ Can be very accurate if assumptions met ○ Can be easily used on active animals Cons: ○ Dependent upon certain assumptions Aerobic metabolism only (eg. known relationship between O2 or CO2) and ATP ○ Must sample gases effectively All relevant gases, limit leaks ○ Animal “tied” to equipment, at least Respiratory Quotient Type of fuel being used can be monitored by measuring the RQ (similar to Respiratory Exhchange Ratio - RER) Respiratory quotient (RQ) = rate of CO2 production/O2 consumption ○ What is actually used/produced by mitochondria ○ RQ values: 0.7 = lipids 1.0 = carbohydrates 0.85 ≈ proteins ○ Catabolism of protein is negligible RQ can directly reveal ration of carb/fat oxidation in such cases RER is measured at respiratory interface (actual breathing animal) - can be ‘uncoupled’ from what’s happening at mitochondria RQ and ATP Turnover If the relative amounts of O2 AND CO2 consumed and produced, respectively, differ depending on which fiel is being oxidized ○ Does # of ATP produced per unit molecular O2 consumed vary as well? ○ How does the ATP/O stoichiometric relationship vary with fuel type? Molecular Stoichiometry of ATP production and O2 Consumption Isolated cells ○ To produce a given # of ATP molecules 14.9% - 18.7% more O2 required when oxidizing fats, compared to carbs Fuel Use And Oxygen Consumption in The Hummingbird If the “cost” of hovering flight (i.e. amount of ATP turnover needed) is constant, but the fuel being oxidized varies… ○ Hypothesis: hovering VO2 will be 15-19% greater when hummingbird is fasted (burning fat) than when fed (burning carbs) ○ Results: Sugar is a more “O2-efficient” fuel